The cytokine IL-4 is the single most important cytokine involved in the development and regulation of humoral immune responses. IL-4 drives the differentiation of naive CD4+ T cells into T helper type 2 (Th2) cells, directs isotype switching in B cells to IgE and IgG4 (IgG1 in mice), and regulates cell growth and death. As such, IL-4 has a pivotal role in regulating inflammatory responses, autoimmune diseases, immune responses to infection, and allergy, including asthma. IL-4 regulates this diverse array of immune functions in part through the activation of specific sets of genes in different cell types. IL-4 induces gene expression by inducing Janus kinase(Jak)-mediated activation of the transcription factor Stat6. Importantly, binding of Stat6 alone to DNA is not sufficient to activate transcription and cooperation of Stat6 with other factors is necessary. Recent evidence suggests that other signaling pathways, including the IRS-2/PI 3-kinase and PKC pathways, may be important for Stat6-dependent gene expression, but how other pathways cooperate with Stat6 is entirely unknown. The long-range goal of this project is to understand how IL-4 signaling regulates gene expression to mediate diverse biological effects. The objective of this application is to elucidate the mechanisms by which the Jak/Stat6 pathway cooperates with other signaling pathways and cellular factors to activate gene expression in response to IL-4. The central hypothesis for the proposed research is that IL-4-responsive gene expression is regulated not only by Jak-mediated tyrosine phosphorylation of Stat6 but also by serine kinase signaling pathways that converge on Stat6 and other required transcription cofactors to promote their cooperative interaction. This hypothesis is supported by our recent demonstration that IL-4 induces serine phosphorylation of Stat6. To test the central hypothesis, this proposal will pursue three specific aims: 1) determine the sites of Star6 serine phosphorylation, the kinetics and stability of serine phosphorylation and the role of IL-4R-mediated signaling events in Stat6 serine phosphorylation; 2) determine the role of IL-4-induced Stat6 serine phosphorylation in the regulation of Stat6-dependent gene expression; and 3) establish the mechanisms by which Stat6 cooperates with other necessary cofactors in the regulation of IL-4-dependent gene expression. Serine phosphorylation sites in Stat6 will be determined by peptide mapping and mass spectrometry. Their role in regulating Stat6 expression and function will be analyzed in Stat6-deficient B cells by retrovirus-mediated transduction. The proposed studies will focus on the regulation of the unrearranged IgG1 immunoglobulin gene in mouse B cells as a model for IL-4-induced gene expression. The proposed studies will significantly advance our current limited understanding of how IL-4 signaling and Stat6 regulate gene expression in lymphocytes as well as in other cell types. A better understanding of how IL-4-mediated signals cooperate to regulate gene expression will also allow development of novel approaches for modulating immune responses and disease processes regulated or influenced by IL-4.